Screening And Study Of Monoclonal Neutralizing Antibodies Against SARS-CoV-2 S Protein Based On ACE2

Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)infection,mediated by the binding of the stinging(S)protein to the human cell surface angiotensin-converting enzyme 2(ACE2)receptor,is widely spread and lacks specificity,The widespread spread of SARS-Co V-2 and the lack of specific therapeutic agents have resulted in tens millions of deaths and posed a significant threat to human health and public safety.Therefore,an urgent clinical need for a drug that can specifically target COVID-19.Based on the fact that SARS-CoV-2 infection of host cells relies on the binding of S protein to ACE2,this project aims to develop high-affinity antibodies using the Receptor Binding Domain(RBD)of S protein as a specific antigenic epitope and to evaluate the performance of the antibodies.Splenocytes from BALB/c mice immunized with SARS-CoV-2 RBD protein were fused with human myeloma cells,and positive hybridoma cells were screened using ELISA with RBD as the antigen.Sixteen monoclonal cell lines that stably secreted anti-SARS-Co V-2 antibody were finally obtained.The m RNA of the monoclonal cell lines was extracted,and the monoclonal antibody sequences were obtained by reverse transcription,PCR,DNA purification,ligation,transformation and sequencing.After synthesizing the plasmids and transient transfection,four monoclonal antibodies with high purity were successfully purified.We further characterized the performance of 14E3 HL and 14F4 HL by focusing on affinity,competitive binding sites,pseudovirus neutralization activity assay,thermal stability assay and pharmacokinetic assay.The results showed that 14F4 HL and 14E3 HL bound to the RBD protein with a Concentration for 50% of maximal effect(EC50)of 0.00253 μg/m L,0.02683μg/m L and 0.0245 μg/m L with the positive control LY-Co V555,JS016,respectively.14F4 HL and 14E3 HL had high affinity for all mutants of RBD except the delta mutant and were able to bind RBD proteins competitively with ACE2.14E3 HL and LY-Co V555 were able to bind RBD competitively with 14F4 HL,considering The Melting Temperature(Tm)values of 14F4 HL and 14E3 HL were both 59.750°C,lower than those of LY-Co V555 and JS016(Etesevimab).14F4 HL and 14E3 HL were effective in neutralizing wild and several other mutant pseudoviruses in vitro,with drug elimination half-lives(T/2)in mice of 153.04 h and 114.64 h,much longer than that of LY-Co V555.14F4 HL had an Area Under Curve(AUC)of 0-192 h,reaching twice that of the positive control LY-Co V555.In conclusion,the two monoclonal antibodies,14F4 HL and 14E3 HL,screened in this study have high affinity for RBD.It can be used as a candidate for monotherapy or cocktail therapy for infections with mutant strains of viruses other than delta mutants.The two monoclonal antibodies have good drug-forming properties and are suitable for further humanisation studies.